A large body of evidence suggests that nitric oxide (NO) gas play a key role in nonadrenergic noncholinergic (NANC) inhibitory neuro-smooth muscle transmission throughout the body including the gut. Clinically, deficiency of nitrergic transmission has been shown to be responsible for many major gastrointestinal motility disorders. However, several important questions regarding nitrergic neurotransmission remain unanswered. For example, the precise site(s) of production of NO at the neuro-smooth muscle junctions, including the prejunctional nerve varicosities, the postjunctional smooth muscle cells or the intramuscular type of interstitial cells of Coal (ICC-IM) remain undefined. Some investigators believe that NO produced in the postjunctional structures is essential in the neuromuscular transmission. It is also unclear how this highly diffusible gas can fulfill the function of a neurotransmitter that requires highly controlled release. The release of a classical neurotransmitter is regulated by exocytosis of a small fraction of the secretory granules that are docked onto the plasma membrane. However, NO is not stored in the secretory granules and is thought to be produced on demand during the neurotransmission. The nerve varicosities that contain nNOS also contain peptide VIP, however, the role VIP in nitrergic neuro smooth muscle neurotransmission is controversial. We propose models of regulation of nNOS and an intermediary role of VIP in nitrergic neurotransmission. To test these models we plan to utilize: (1) a novel technique using fluorescent dye markers and multi-photon microscopy to visualize NO and calcium signals in real-time in live nerve varicosities and other structures at the neuromuscular junctions; (2) immunocytological studies using multi-photon microscopy to localize the proteins that may be involved in cycling of nNOS from catalytically inactive to catalytically active forms; (3) intracellular recordings of the nitrergic inhibitory junction potentials. Using the above techniques, we plan to study the effects of pharmacological manipulations to test the proposed models. These studies will help define:(1) whether during the neurotransmission NO is produced in the nerve varicosities and also in the postjunctional smooth muscle cells or ICC-IM; (2) the regulation of nNOS that permits NO gas to function as a neurotransmitter; (3) the intermediary role of VIP and its unique receptor subtype in nitrergic neurotransmission. This information may help in understanding physiology, and the pathogenesis and rational treatment of clinical disorders due to deficiency of nitrergic neuro-smooth muscle transmission.